1. Field Of The Invention
This invention relates to protein-enzyme complexes in membrane form and more particularly to enzymatically active protein-enzyme complex membranes which can be used for catalyzing enzymatic reactions. In another aspect, this invention relates to methods for preparing said enzymatically active membranes and to methods of using said enzymatically active membranes.
2. Description Of The Prior Art
Enzymes are protein catalysts which have been used for a wide variety of industrial and research applications, particularly in pharmaceuticals, paper and textile processing, etc. They are highly specific in their activity and generally do not generate significant quantities of undesirable byproducts. Enzyme reactions are industrially advantageous since they do not require a large investment in heat transfer equipment and can be easily staged, thereby minimizing the problems associated with interstage product separations.
One problem which has long concerned those dealing with industrial applications of enzymes, however, is the difficulty in separating or recovering enzyme materials. In most commerical processes, the enzymatic reaction is effected by simply admixing the enzyme with the substrate, and thereafter inactivating and/or recovering the enzyme from the products or the unreacted substrate following the reaction. This procedure, however, has frequently resulted in damage to the product and inherent loss of large quantities of enzyme, since usually no enzyme is recovered or, if attempted, the yields are quite low.
Another problem which has been of significant concern to those engaged in this technology, is that the enzymes usually are used in an aqueous dispersion form. As a rule, however, enzymes in this form have a limited shelf life and, especially, if stored in dilute form, will undergo rapid loss of activity upon storage.
To alleviate these problems, the art has developed various so-called "immobilized enzymes" in which the enzymes are immobilized or bound to inert or insoluble carriers. At the completion of the enzymatic reaction, these insoluble enzyme-containing materials can be separated from the unreacted substrate or product by techniques such as ultrafiltration or the like.
The selection of a suitable inert carrier, however, has been quite difficult, since the carrier must not only be inert to the enzyme, but it must not inhibit the catalytic activity of the enzyme, nor cause undesirable unspecific adsorption. Moreover, the carrier should present a minimum of steric hindrance toward the enzyme-substrate reaction. A wide variety of prior art carriers have been proposed, depending upon the particular type of enzyme used and the particular enzymatic reaction desired. For instance, among those prior art carriers disclosed in the open literature include, synthetic polymers such as polyamides, cellulose derivatives, various clays, and ion-exchange resins, particularly DEAE-cellulose, and DEAE-dextrans, as discussed in Suzuki, et al., Agr. Biol. Chem., Volume 30, No. 8, Pages 807-812 (1966). Prior art methods of preparing immobilized enzymes have included direct covalent bonding, indirect bonding through an intermediate compound, cross-linking of the enzyme or trapping the enzyme in polymer lattices.
None of these prior art techniques or carriers, however, have been entirely satisfactory for all purposes. Synthetic polymer carriers are expensive and frequently are not readily available. Moreover, they often require special treatment in order to chemically bind the enzyme to the carrier. The cellulose derivatives are generally unsuitable as binders for carbohydrases, since carbohydrates are substrates for these enzymes, Ion-exchange resins, such as DEAE-cellulose and DEAE-dextran, have ion-exchange properties, which may not be desirable for certain applications. The problem of enzyme liberation from a carrier is one weak point in many immobilized enzyme preparations, and is particularly troublesome in the case of amylase bound to acid clay, which becomes liberated during the hydrolytic reaction of starch.
One particular disadvantage of the prior art methods of immobilizing enzymes is that they have resulted in the formation of insoluble pastes, particles or granular materials. While such forms are suitable, and even possibly desirable for certain applications, for other applications, these forms impose severe limitations, especially when they are used for large-scale or long-term continuous processes.
A need exists, therefore, for an enzyme carrier which can be formed into a variety of shapes and hence can be used as a structural part of a reaction system, so as to eliminate entirely separation problems. More specifically, a need exists for a membrane or film-like carrier which is capable of complexing and binding enzymes thereto without hindering their catalytic activity, so that enzymatic reactions can be effected merely by passing the substrate over the active membrane or film. The present invention fills such a need.